Melt-electrowriting-enabled anisotropic scaffolds loaded with valve interstitial cells for heart valve tissue Engineering.

Tissue engineered heart valves (TEHVs) demonstrates the potential for tissue growth and remodel, offering particular benefit for pediatric patients. A significant challenge in designing functional TEHV lies in replicating the anisotropic mechanical properties of native valve leaflets. To establish a biomimetic TEHV model, we employed melt-electrowriting (MEW) technology to fabricate an anisotropic PCL scaffold. By integrating the anisotropic MEW-PCL scaffold with bioactive hydrogels (GelMA/ChsMA), we successfully crafted an elastic scaffold with tunable mechanical properties closely mirroring the structure and mechanical characteristics of natural heart valves. This scaffold not only supports the growth of valvular interstitial cells (VICs) within a 3D culture but also fosters the remodeling of extracellular matrix of VICs. The in vitro experiments demonstrated that the introduction of ChsMA improved the hemocompatibility and endothelialization of TEHV scaffold. The in vivo experiments revealed that, compared to their non-hydrogel counterparts, the PCL-GelMA/ChsMA scaffold, when implanted into SD rats, significantly suppressed immune reactions and calcification. In comparison with the PCL scaffold, the PCL-GelMA/ChsMA scaffold exhibited higher bioactivity and superior biocompatibility. The amalgamation of MEW technology and biomimetic design approaches provides a new paradigm for manufacturing scaffolds with highly controllable microstructures, biocompatibility, and anisotropic mechanical properties required for the fabrication of TEHVs.

Chimeric Protein Template-Induced Shape Control of Bone Mineral Nanoparticles and Its Impact on Mesenchymal Stem Cell Fate.

Protein-mediated molecular self-assembly has become a powerful strategy to fabricate biomimetic biomaterials with controlled shapes. Here we designed a novel chimeric molecular template made of two proteins, silk fibroin (SF) and albumin (ALB), which serve as a promoter and an inhibitor for hydroxyapatite (HA) formation, respectively, to synthesize HA nanoparticles with controlled shapes. HA nanospheres were produced by the chimeric ALB-SF template, whereas HA nanorods were generated by the SF template alone. The success in controlling the shape of HA nanoparticles allowed us to further study the effect of the shape of HA nanoparticles on the fate of rat mesenchymal stem cells (MSCs). We found that the nanoparticle shape had a crucial impact on the cellular uptake and HA nanospheres were internalized in MSCs at a faster rate. Both HA nanospheres and nanorods showed no significant influence on cell proliferation and migration. However, HA nanospheres significantly promoted the osteoblastic differentiation of MSCs in comparison to HA nanorods. Our work suggests that a chimeric combination of promoter and inhibitor proteins is a promising approach to tuning the shape of nanoparticles. It also sheds new light into the role of the shape of the HA nanoparticles in directing stem cell fate.

Integrated polycaprolactone microsphere-based scaffolds with biomimetic hierarchy and tunable vascularization for osteochondral repair.

Osteochondral lesion potentially causes a variety of joint degenerative diseases if it cannot be treated effectively and timely. Microfracture as the conservative surgical choice achieves limited results for the larger defect whereas cartilage patches trigger integrated instability and cartilage fibrosis. To tackle aforementioned issues, here we explore to fabricate an integrated osteochondral scaffold for synergetic regeneration of cartilage and subchondral bone in one system. On the macro level, we fabricated three integrated scaffolds with distinct channel patterns of Non-channel, Consecutive-channel and Inconsecutive-channel via Selective Laser Sintering (SLS). On the micro level, both cartilage zone and subchondral bone zone of integrated scaffold were made of small polycaprolactone (PCL) microspheres and large PCL microspheres, respectively. Our findings showed that Inconsecutive-channel scaffolds possessed integrated hierarchical structure, adaptable compression strength, gradient interconnected porosity. Cartilage zone presented a dense phase for the inhibition of vessel invasion while subchondral bone zone generated a porous phase for the ingrowth of bone and vessel. Both cartilage regeneration and subchondral bone remodeling in the group of Inconsecutive-channel scaffolds have been demonstrated by histological evaluation and immunofluorescence staining in vivo. Consequently, our current work not only achieves an effective and regenerative microsphere scaffold for osteochondral reconstruction, but also provides a feasible methodology to recover injured joint through integrated design with diverse hierarchy. STATEMENT OF SIGNIFICANCE: Recovery of osteochondral lesion highly depends on hierarchical architecture and tunable vascularization in distinct zones. We therefore design a special integrated osteochondral scaffold with inconsecutive channel structure and vascularized modulation. The channel pattern impacts on mechanical strength and the infiltration of bone marrow, and eventually triggers synergetic repair of osteochondral defect. The cartilage zone of integrated scaffolds consisted of small PCL microspheres forms a dense phase for physical restriction of vascularized infiltration whereas the subchondral bone zone made of large PCL microspheres generates porous trabecula-like structure for promoting vascularization. Consequently, the current work indicates both mechanical adaptation and regional vascularized modulation play a pivotal role on osteochondral repair.

Mitochondria-Targeted BODIPY Nanoparticles for Enhanced Photothermal and Photoacoustic Imaging In Vivo.

Short-wavelength absorption and emission (<600 nm), hydrophobicity, and low selectivity have greatly restricted the biomedical applications of BODIPY. Herein, a series of mitochondria-targeted BODIPY nanoparticles with a cationic triphenylphosphine (TPP) group (Mito-BDP1-5 NPs) bearing different lengths of ethylene glycol (0-4 units), along with HO-BDP5 without a cationic TPP group for comparison, have been rationally designed and prepared to investigate the interplay between their structures and the related properties. Our studies found that Mito-BDP1-4 NPs showed a tendency of aggregation and precipitation while Mito-BDP5 NPs could be stable in aqueous solutions. Compared with HO-BDP5, tailor-made Mito-BDP5 possessed a high photothermal conversion efficiency (PCE) of 76.6 vs 9.0% and exhibited the highest photoinduced cytotoxicity. Upon NIR irradiation, the temperature induced by Mito-BDP5 NPs increased rapidly from room temperature to 76.0 °C in vitro and 50.0 °C at the tumor site in vivo within 5 min. Furthermore, effective mitochondrial imaging in vitro, photothermal imaging (PTI), and photoacoustic imaging (PAI) in vivo were achieved. In this paper, we developed tailor-made photothermal agents for targeting mitochondria and enhancing the PTI and PAI performances, which could be potentially applied in clinical precision theranostics.

Progenitor cell-derived exosomes endowed with VEGF plasmids enhance osteogenic induction and vascular remodeling in large segmental bone defects.

Large segmental bone regeneration remains a great challenge due to the lack of vascularization in newly formed bone. Conventional strategies primarily combine bone scaffolds with seed cells and growth factors to modulate osteogenesis and angiogenesis. Nevertheless, cell-based therapies have some intrinsic issues regarding immunogenicity, tumorigenesis, bioactivity and off-the-shelf transplantation. Exosomes are nano-sized (50-200 nm) extracellular vesicles with a complex composition of proteins, nucleic acids and lipids, which are attractive as therapeutic nanoparticles for disease treatment. Exosomes also have huge potential as desirable drug/gene delivery vectors in the field of regenerative medicine due to their excellent biocompatibility and efficient cellular internalization. Methods: We developed a cell-free tissue engineering system using functional exosomes in place of seed cells. Gene-activated engineered exosomes were constructed by using ATDC5-derived exosomes to encapsulate the VEGF gene. The specific exosomal anchor peptide CP05 acted as a flexible linker and effectively combined the engineered exosome nanoparticles with 3D-printed porous bone scaffolds. Results: Our findings demonstrated that engineered exosomes play dual roles as an osteogenic matrix to induce the osteogenic differentiation of mesenchymal stem cells and as a gene vector to controllably release the VEGF gene to remodel the vascular system. In vivo evaluation further verified that the engineered exosome-mediated bone scaffolds could effectively induce the bulk of vascularized bone regeneration. Conclusion: In our current work, we designed specifically engineered exosomes based on the requirements of vascularized bone repair in segmental bone defects. This work simultaneously illuminates the potential of functional exosomes in acellular tissue engineering.

Green Gas-Mediated Cross-Linking Generates Biomolecular Hydrogels with Enhanced Strength and Excellent Hemostasis for Wound Healing.

Forming biomolecular hydrogels with a combination of high strength and biocompatibility is still a challenge. Herein, we demonstrated a green gas (CO2)-mediated chemical cross-linking strategy that can produce a double-network cellulose/silk fibroin hydrogel (CSH) with significantly elevated mechanical strength while bypassing the toxicity of routine cross-linking agents. Specifically, cellulose and silk fibroin (SF) were first covalently cross-linked in NaOH/urea solution to create the primary network. Then, CO2 gas was introduced into the resultant CSH precursor gels to form carbonates to reduce the pH value of the intra-hydrogel environment from basic to neutral conditions. The pH reduction induced the ordered aggregation of cellulose chains and concomitant hydrogen bonding between these chains, leading to the formation of hydrogels with significantly improved mechanical strength. The CSHs could promote the adhesion and proliferation of the mouse fibroblast cell line (L929), and the CSHs proved to be of low hemolysis and could accelerate blood clotting and decrease blood loss. The CSHs with SF content of 1 wt % healed the wound in vivo within only 12 days through the acceleration of re-epithelialization and revascularization. Consequently, our current work not only reported a feasible alternative for wound dressings but also provided a new green gas-mediated cross-linking strategy for generating mechanically strong, hemostatic, and biocompatible hydrogels.

Enhanced effect of nano-monetite hydrosol on dentin remineralization and tubule occlusion.

OBJECTIVES: We aim to investigate the dentin tubule occlusion and remineralization potential of a novel nano-monetite hydrosol (nMH). METHODS: First, nano-monetite hydrosol (nMH) was fabricated by homogeneous precipitation method. Then, the effectiveness of toothpaste with nMH on improving remineralization was evaluated by the measurement of tubule occluding ratio and acid-resistant stability compared with dentifrices comprising nano-hydroxyapatite hydrosol (nHH) and bioactive glass (BG). To explain this result, we studied the ions releasing and remineralization based on gelatin scaffold among nMH, nHH and BG. Finally, the cytotoxicity of these three minerals on Human dental pulp stem cells (HDPSCs) was evaluated. RESULTS: Processing for more than 7 days, the toothpaste containing nMH exhibited the significant remineralization potential and acid-resistant compared with two commercial de-sensitive dentifrices comprising nHH and BG. In addition, cytotoxicity test resulted that nMH has good cell compatibility to HDPSCs below extracts concentration of 3.12mg/mL. SIGNIFICANCE: Small size, the release of Ca2+ and PO43- with high concentration, strongly binding on dental surface, and fast transformation to HAp, were all needed in the preparation of effective dentin tubule occluding biomaterials.

Untangling the co-effects of oriented nanotopography and sustained anticoagulation in a biomimetic intima on neovessel remodeling.

Constructing a small-diameter artificial blood vessel with biological functions and mechanical compliance comparable to native tissues is still a major challenge in vascular tissue engineering. To address the issues of severe thrombosis and unsatisfactory long-term patency in small-diameter vascular grafts, herein we designed a specifically biomimetic intima with an oriented nanotopographical structure and covalently immobilized anticoagulant molecules. The mixture of heparinized silk fibroin (SF-Hep) and polycaprolactone (PCL) was used to produce oriented inner layer and pure PCL was used to fabricate vertically porous outer layer by a two-step cross-electrospinning. Our findings showed that the immobilized heparin significantly influenced adherence and activation of platelets while the oriented nanotopography mainly manipulated the elongation and aligned growth of endothelial cells as well as hemodynamics of blood flow. More importantly, two factors of the oriented structure and anticoagulation presented the obviously synergistic effects on rapid endothelialization, long-term patency and remodeling of neovessel. Consequently, the current study successfully combined biochemical induction of heparin molecule and biophysical stimulation of oriented nanotopography to create an off-the-shelf small-diameter vascular graft with excellent antithrombosis in the early stage and long-term patency in the late stage.

Bioenergetic-active materials enhance tissue regeneration by modulating cellular metabolic state.

Cellular bioenergetics (CBE) plays a critical role in tissue regeneration. Physiologically, an enhanced metabolic state facilitates anabolic biosynthesis and mitosis to accelerate regeneration. However, the development of approaches to reprogram CBE, toward the treatment of substantial tissue injuries, has been limited thus far. Here, we show that induced repair in a rabbit model of weight-bearing bone defects is greatly enhanced using a bioenergetic-active material (BAM) scaffold compared to commercialized poly(lactic acid) and calcium phosphate ceramic scaffolds. This material was composed of energy-active units that can be released in a sustained degradation-mediated fashion once implanted. By establishing an intramitochondrial metabolic bypass, the internalized energy-active units significantly elevate mitochondrial membrane potential (ΔΨm) to supply increased bioenergetic levels and accelerate bone formation. The ready-to-use material developed here represents a highly efficient and easy-to-implement therapeutic approach toward tissue regeneration, with promise for bench-to-bedside translation.

A high-strength biodegradable thermoset polymer for internal fixation bone screws: Preparation, in vitro and in vivo evaluation.

Thermoset polymers synthesized from the polycondensation of glycerol with biocompatible diacids represent a promising class of absorbable materials for biomedical applications. However, the utility of these polymers for bone fixation devices is hampered due to the lack of mechanical strength. Herein we synthesized a high-strength thermoset polymer, poly(glycerol-succinate) (PGS), via a catalyst-free and solvent-free reaction. The bending strength of PGS reaches 122.01 ±â€¯8.82 MPa, signifying its great potential for fixation devices. The degradation property of the polymer can be tuned by adjusting the monomer ratio and reaction time. Bone screws based on the PGS polymer were successfully manufactured using a lathe. In vitro evaluation showed the PGS polymer was able to well support cell adhesion and proliferation. In vivo evaluation using a rat subcutaneous implantation model showed that the inflammatory response to the polymer was mild. After the PGS screws were implanted in the rabbit femoral condyle for 12 weeks, micro-computed tomography (micro-CT) and histological analysis revealed that the screws achieved good osseointegration. Consequently, the polymer developed in current study can serve as internal fixation devices due to the proper mechanical strength, excellent biocompatibility, and feasibility of manufacturing screws.

Poly(amino acid)-Based Carrier for Drug Delivery Systems.

Micro/nanoscale carrier is a crucial component of the drug delivery system that enables to be well designed for improving the pharmacological and therapeutic properties of drug administration. Herein we systemically review a special vector of natural poly(amino acid) (PAA) that has been extensively applied in the controlled delivery of drug, protein and gene due to its excellent biocompatibility, controlled degradability, and flexible physicochemical modification. Natural PAA is a group of poly(ionic) molecules that present various biological roles and putative technical functions. The intention of this review is to thoroughly summarize three classic types of PAA involving anionic, cationic and neutral PAA as well as their applications on drug delivery systems.

Selective laser sintering scaffold with hierarchical architecture and gradient composition for osteochondral repair in rabbits.

Osteochondral defects cannot be adequately self-repaired due to the presence of the sophisticated hierarchical structure and the lack of blood supply in cartilage. Thus, one of the major challenges remaining in this field is the structural design of a biomimetic scaffold that satisfies the specific requirements for osteochondral repair. To address this hurdle, a bio-inspired multilayer osteochondral scaffold that consisted of the poly(ε-caprolactone) (PCL) and the hydroxyapatite (HA)/PCL microspheres, was constructed via selective laser sintering (SLS) technique. The SLS-derived scaffolds exhibited an excellent biocompatibility to support cell adhesion and proliferation in vitro. The repair effect was evaluated by implanting the acellular multilayer scaffolds into osteochondral defects of a rabbit model. Our findings demonstrated that the multilayer scaffolds were able to induce articular cartilage formation by accelerating the early subchondral bone regeneration, and the newly formed tissues could well integrate with the native tissues. Consequently, the current study not only achieves osteochondral repair, but also suggests a promising strategy for the fabrication of bio-inspired multilayer scaffolds with well-designed architecture and gradient composition via SLS technique.

Microsphere-based selective laser sintering for building macroporous bone scaffolds with controlled microstructure and excellent biocompatibility.

Fabrication of bulk biomaterials with controlled structures and excellent properties is increasingly important in tissue engineering, but remains a major challenge in the current stage. Herein we used selective laser sintering (SLS) to construct a series of three-dimensional (3D) bone scaffolds with uniform multi-scaled porosity, moderate mechanical properties as well as good biocompatibility. As starting architectural units for SLS, the pure microspheres of polycaprolactone (PCL) and the composite microspheres of PCL and hydroxyapatite (HA) were firstly synthesized via a modified solvent evaporation method, respectively. Our findings showed that the as-prepared microspheres exhibited the uniform size and monodispersity. Moreover, the microsphere-based 3D scaffolds generated by SLS technique showed a multi-scaled porous structure, and adequate mechanical features. Both in vitro and in vivo evaluations further demonstrated that the resultant SLS-derived scaffolds can not only manipulate multiple stem cell behaviors including promoting cell adhesion, supporting cell proliferation and inducing cell differentiation in vitro, but also showed an excellent histocompatibility and induced the vascularization of newly formed tissue in vivo. Consequently, our current study suggests a feasible and effective protocol for fabricating new biomimetic bone biomaterials via SLS technique, also paves a new way for other bulk biomaterials.

Biomimetic self-assembly of apatite hybrid materials: from a single molecular template to bi-/multi-molecular templates.

The self-assembly of apatite and proteins is a critical process to induce the formation of the bones and teeth in vertebrates. Although hierarchical structures and biomineralization mechanisms of the mineralized tissues have been intensively studied, most researches focus on the self-assembly biomimetic route using one single-molecular template, while the natural bone is an outcome of a multi-molecular template co-assembly process. Inspired by such a mechanism in nature, a novel strategy based on multi-molecular template co-assembly for fabricating bone-like hybrid materials was firstly proposed by the authors. In this review article we have summarized the new trends from single-molecular template to bi-/multi-molecular template systems in biomimetic fabrication of apatite hybrid materials. So far, many novel apatite hybrid materials with controlled morphologies and hierarchical structures have been successfully achieved using bi-/multi-molecular template strategy, and are found to have multiple common features in comparison with natural mineralized tissues. The carboxyl, carbonyl and amino groups of the template molecules are identified to initiate the nucleation of calcium phosphate during the assembling process. For bi-/multi-molecular templates, the incorporation of multiple promotion sites for calcium and phosphate ions precisely enables to regulate the apatite nucleation from the early stage. The roles of acidic molecules and the synergetic effects of protein templates have been significantly recognized in recent studies. In addition, a specific attention is paid to self-assembling of apatite nanoparticles into ordered structures on tissue regenerative scaffolds due to their promising clinical applications ranging from implant grafts, coatings to drug and gene delivery.

Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds.

A virus-activated matrix is developed to overcome the challenge of forming vascularized bone tissue. It is generated by filling a 3D printed bioceramic scaffold with phage nanofibers displaying high-density RGD peptide. After it is seeded with mesenchymal stem cells (MSCs) and implanted into a bone defect, the phage nanofibers induce osteogenesis and angiogenesis by activating endothelialization and osteogenic differentiation of MSCs.

Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells.

Hydroxyapatite (HA), the principal component of bone mineral, shows osteoconductive properties when employed for coating metal implants as well as scaffold materials in synthetic bone grafts. With the goal of providing this material with osteoinductive capabilities to promote faster bone regeneration, we show an easy approach to functionalize HA implant surfaces and enrich them with osteoinductive properties by the use of HA-binding modular peptides. The modular peptides are designed as a combination of two domains, an HA-binding peptide motif and an osteogenic peptide motif derived from the osteogenic growth peptide (OGP) or bone morphometric protein 7 (BMP-7). To identify the best HA-binding peptide, several nature-inspired peptides derived from natural bone extracellular matrix proteins (bone sialoprotein, osteonectin, osteocalcin, and salivarin statherin) were compared for HA-binding activity, revealing concentration-dependent and incubation-time-dependent behaviours. We discovered that a Poly-E heptamer (E7) is the best HA-binding peptide, and thus combined it with a second osteogenic peptidic domain to create an osteoinductive modular peptide. After binding/release characterization, we found that the addition of the second osteogenic peptide domain did not change the binding profile of the modular peptides and caused only a slight change in their release kinetics. Mesenchymal stem cells (MSCs) were cultured on the HA substrates functionalized with modular peptides, and cell adhesion, proliferation, and differentiation in a basal medium (i.e., without any osteogenic supplements) were investigated. Gene expression data clearly showed that MSCs were committed to differentiate into osteoblasts in the presence of the modular peptides. HA discs functionalized with the E7 BMP-7 modular peptide showed the best capability in inducing the osteogenic differentiation of MSCs among all modular peptides studied. The modular peptides can easily be used to functionalize the HA implants through its constituent HA-binding motif, leaving the osteogenic peptide motif protruding from the surface for inducing osteogenesis. Our work opens up a new approach to the formulation of new bioactive HA coatings and implants for bone and dental repair.

Osteogenic differentiation of bone marrow mesenchymal stem cells on the collagen/silk fibroin bi-template-induced biomimetic bone substitutes.

Biomimetic bone substitutes of collagen-silk fibroin/hydroxyapatite (COL-SF/HA) were synthesized via a bi-template-induced coassembly strategy. Collagen-hydroxyapatite (COL-HA) and silk fibroin-hydroxyapatite (SF-HA) served as controls were prepared with similar method. The osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) seeded onto the resulting materials was evaluated both in vitro and in vivo. The results suggested that the bi-template-induced biomimetic substitutes were able to support the growth and proliferation of BMSCs. We further demonstrated that BMSCs were stimulated to differentiate into the osteoblast cell lineage by evaluating several specific osteogenic markers including staining of alkaline phosphate (ALP) and calcium nodular and expression of osteogenic genes of osteocalcin (OCN) and osteonectin (ONN). The rat femoral defect model was used to assess the aforementioned biomimetic bone substitutes combined with BMSCs in vivo. Histological analysis indicated that the bi-template material exhibited good biocompatibility and strong ability of the new bone formation in comparison with the control of single-template material in vivo.

Dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan.

Heparinized biomaterials exhibit great anticoagulant properties. However, they promote proliferation of Staphylococcus aureus (S. aureus) and therefore cause infection within the bloodstream upon implantation in vivo. In the present study, an interesting dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan was synthesized. First, heparin was grafted onto the silk fibroin by covalent immobilization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). All data gathered from Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and elemental analysis (EA) indicated that the heparin was successfully immobilized onto the silk fibroin. The dual-functional composite of heparinized silk fibroin and chitosan was then fabricated by a blending method. The anticoagulant activity of the heparinized materials was evaluated using the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). The results showed that both heparinized silk fibroin and the composite material exhibited better hemocompatibility in comparison with single silk fibroin or chitosan. The antibacterial property of the materials was investigated by the pour-plate method. Results further suggested that the composite antibacterial property with respect to S. aureus was significantly enhanced. The dual-functionality of the composite material may supply a potential choice in blood contact devices.

Collagen/silk fibroin bi-template induced biomimetic bone-like substitutes.

A novel bi-template induced co-assembly method was employed to fabricate biomimetic bone substitute materials, collagen (COL)-fibroin/hydroxyapatite (COL-SF/HA) composite by using a combination of Type I COL and silk fibroin (SF) molecular templates. As a control, COL/HA and SF/HA composites were also synthesized via single-template assembly technology. The structure and morphology of the resulting assembly composites were investigated by X-ray diffractometer, Fourier transform infrared spectra, transmission electron microscope, and thermogravimetric analysis. Their sizes and size distributions were measured by DLS. The results indicated that the mineral phases in COL-SF/HA, COL-HA, and SF-HA composites were needle-like nano-HA crystals. In comparison to those in COL-HA and SF-HA, the mineral phase in COL-SF/HA displayed smaller size and more narrow distribution. Of all above biomimetic composites, the HA was well assembled with molecular template(s), and the organic content of the composite was about 12%-20%, which was quite similar to the natural bone in composition. CD and SDS-PAGE were used to examine the secondary structure and subunit composition of template proteins. The results revealed that the spatial structure of co-assembly template proteins played a pivotal role in controlling and regulating HA crystal nucleation and growth. Based on the experimental results above, a possible co-assembly mechanism for the HA growing on fibrous bi-template proteins was suggested.